For a number of years lipolytic enzymes have been used in detergents to remove lipid or fatty stains from clothes and other textiles.
For instance, various microbial lipases have been suggested as detergent enzymes. Examples of such lipases include a Humicola lanuginosa lipase, e.g., described in EP 258,068 and EP 305,216, a Rhizomucor miehei lipase, e.g., as described in EP 238,023, a Candida lipase, such as a C. antarctica lipase., e.g., the C. antarctica lipase A or B described in EP 214,761, a Pseudomonas lipase such as a P. alcaligenes and P. pseudoalcaligenes lipase, e.g., as described in EP 218,272, a P. cepacia lipase, e.g., as described in EP 331,376, a Bacillus lipase, e.g., a B. subtilis lipase (Dartois et al., Biochemica et Biophysica Acta 1131, pp. 253-260 (1993)), a B. stearothermophilus lipase (JP 64/744992) and a B. pumilus lipase (EP 91 00664).
Furthermore, a number of cloned lipases have been described, including the Penicillium camembertii lipase described by Yamaguchi et al., Gene 103, pp. 61-67 (1991), the Geotricum candidum lipase (Shimada et al., J. Biochem. 106, 383-88 (1989)), and various Rhizopus lipases such as a R. delemar lipase (Hass et al., Gene 109, pp. 107-13 (1991)), a R. niveus lipase (Kugimiya, Biosci. Biotech. Biochem. 56, pp. 716-19 (1992)), and a R. oryzae lipase.
The primary structure of a number of lipases has been determined and described in the literature (Boel et al., Lipids 23, pp 701-06 (1988), de Caro et al., Biochim. Biophys. Acta 671, pp. 129-38 (1981), Winkler et al., Nature 343, pp. 771-74 (1990)). Furthermore, the tertiary structure of a more limited number of lipases has been elucidated (Brady et al., Nature 343, 767-70 (1990) and Schrag et al., i Nature 351, pp. 761-64 (1991)). From these investigations it appears that lipases seem to have certain structural features in common, but that, on the other hand, major structural variations also exist among the lipases.
Other types of lipolytic enzymes include cutinases, e.g., a cutinase derived from Pseudomonas mendocina (WO 88/09367), or from Fusarium solani pisi (WO 90/09446).
In recent years attempts have been made to prepare lipase variants having improved properties for detergent purposes.
PCT/DK93/00225 describes lipase variants with improved properties, in which an amino acid residue occupying a critical position of the lipase has been modified.
EP 407,225 discloses lipase variants with improved resistance towards proteolytic enzymes, which have been prepared by specifically defined amino acid modifications.
EP 260,105 describe hydrolases in which an amino acid residue within 15 .ANG. from the active site has been substituted.
All of the above mentioned lipase variants have been constructed by use of site-directed mutagenesis resulting in a modification of specific amino acid residues which have been chosen either on the basis of their type or on the basis of their location in the secondary or tertiary structure of the parent lipase.
An alternative approach for constructing mutants or variants of a given protein has been based on random mutagenesis. For instance, U.S. Pat. No. 4,898,331 and WO 93/01285 disclose such techniques.
It is an object of the present invention to prepare lipolytic enzymes having improved washing and/or dishwashing properties.